1.1 Field of the Invention
The present invention relates generally to the fields of molecular biology. Methods and compositions comprising DNA sequences, and polypeptides derived from Bacillus thuringiensis for use in insecticidal formulations and the development of transgenic insect-resistant plants are provided. Novel nucleic acids obtained from Bacillus thuringiensis that encode coleopteran-toxic polypeptides are disclosed. Various methods for making and using these nucleic acids, synthetically modified DNA sequences encoding tIC851 polypeptides, and native and synthetic polypeptide compositions are also disclosed. The use of DNA sequences as diagnostic probes and templates for protein synthesis, and the use of polypeptides, fusion proteins, antibodies, and peptide fragments in various insecticidal, immunological, and diagnostic applications are also disclosed, as are methods of making and using nucleic acid sequences in the development of transgenic plant cells comprising the polynucleotides.
1.2 Description of the Related Art
Environmentally-sensitive methods for controlling or eradicating insect infestation are desirable in many instances, in particular when crops of commercial interest are at issue. The most widely used environmentally-sensitive insecticidal formulations developed in recent years have been composed of microbial pesticides derived from the bacterium Bacillus thuringiensis. B. thuringiensis is well known in the art, and is characterized morphologically as a Gram-positive bacterium that produces crystal proteins or inclusion bodies which are aggregations of proteins specifically toxic to certain orders and species of insects. Many different strains of B. thuringiensis have been shown to produce insecticidal crystal proteins. Compositions including B. thuringiensis strains which produce insecticidal proteins have been commercially-available and used as environmentally-acceptable insecticides because they are quite toxic to the specific target insect, but are harmless to plants and other non-targeted organisms.
There are several toxin categories established based on primary structure information and the degree of toxin similarities to another. Over the past decade research on the structure and function of B. thuringiensis toxins has covered all of the major toxin categories, and while these toxins differ in specific structure and function, general similarities in the structure and function are assumed. Based on the accumulated knowledge of B. thuringiensis toxins, a generalized mode of action for B. thuringiensis toxins has been created and includes: ingestion by the insect, solubilization in the insect midgut (a combination stomach and small intestine), resistance to digestive enzymes sometimes with partial digestion actually xe2x80x9cactivatingxe2x80x9d the toxin, binding to the midgut cells, formation of a pore in the insect cells and the disruption of cellular homeostasis (English and Slatin, 1992).
Many of the xcex4-endotoxins are related to various degrees by similarities in their amino acid sequences. Historically, the proteins and the genes which encode them were classified based largely upon their spectrum of insecticidal activity. The review by Schnepf et al. (Microbiol. Mol. Biol. Rev. (1998) 62:775-806) discusses the genes and proteins that were identified in B. thuringiensis prior to 1998, and sets forth the most recent nomenclature and classification scheme as applied to B. thuringiensis insecticidal genes and proteins. Using older nomenclature classification schemes, cry1 genes were deemed to encode lepidopteran-toxic Cry1 proteins, cry2 genes were deemed to encode Cry2 proteins toxic to both lepidopterans and dipterans, cry3 genes were deemed to encode coleopteran-toxic Cry3 proteins, and cry4 genes were deemed to encode dipteran-toxic Cry4 proteins. However, new nomenclature systematically classifies the Cry proteins based upon amino acid sequence homology rather than upon insect target specificities. The classification scheme for many known toxins, not including allelic variations in individual proteins, including dendograms and full Bacillus thuringiensis toxin lists is summarized and regularly updated by the B. thuringiensis Pesticidal Crystal Protein Nomenclature Committee which will periodically publish a comprshensive list of B.t. toxins which will also be available through the internet as described in Crickmore et al., Microbiol. Mol. Biol. Rev. 62:807-813 (1989).
Most of the nearly 200 Bt crystal protein toxins presently known have some degree of lepidopteran activity associated with them. The large majority of Bacillus thuringiensis insecticidal proteins which have been identified do not have coleopteran controlling activity. Therefore, it is particularly important at least for commercial purposes to identify additional coleopteran specific insecticidal proteins.
Cry3 proteins generally display coleopteran activity, however, these generally have limited host range specificity and are not significantly toxic to target pests unless ingested in very high doses. The cloning and expression of the cry3Bb gene has been described (Donovan et al., 1992). This gene codes for a protein of 74 kDa with activity against Coleopteran insects, particularly the Colorado potato beetle (CPB) and the southern corn root worm (SCRW). Improved Cry3Bb proteins have been engineered which display increased toxicity at the same or lower doses than the wild type protein (U.S. Pat. No. 6,023,013; Feb. 8, 2000).
A B. thuringiensis strain, PS201T6, was reported to have activity against WCRW (Diabrotica virgifera virgifera) (U.S. Pat. No. 5,436,002). This strain also had activity against Musca domestica, Aedes aegypti, and Liriomyza trifoli. The vip1A gene, which produces a vegetative, soluble, insecticidal protein, has been cloned and sequenced (Intl. Pat. Appl. Pub. No. WO 96/10083, 1996). This gene produces a protein of approximately 80 kDa with activity against both WCRW and Northern Corn Root Worm (NCRW). Another toxin protein with activity against coleopteran insects, including WCRW, is Cry1Ia, an 81-kDa polypeptide, the gene encoding which has been cloned and sequenced (Intl. Pat. Appl. Pub. No. WO 90/13651, 1990).
The polypeptide of the present invention and the novel DNA sequences that encode the protein represent a new B. thuringiensis crystal protein and gene, and share only insubstantial sequence homology with any previously identified coleopteran inhibitory endotoxins described in the prior art. Similarly, the B. thuringiensis strains of the present invention comprise novel gene sequences that express a polypeptide having insecticidal activity against coleopteran insects, the cotton boll weevil (Anthonomus grandis Boheman) in particular.
Disclosed and claimed herein is an isolated Bacillus thuringiensis xcex4-endotoxin polypeptide comprising SEQ ID NO:8. The inventors have identified an insecticidally-active polypeptide comprising the 632 amino acid long sequence of SEQ ID NO:8 which displays insecticidal activity against coleopteran insects. For example, the inventors have shown that a xcex4-endotoxin polypeptide comprising the sequence of SEQ ID NO:8 has insecticidal activity against boll weevil larvae (BWV), but not against western corn rootworm larvae.
The polypeptide of SEQ ID NO:8 is encoded by a nucleic acid segment comprising at least the open reading frame as shown in SEQ ID NO:7 from nucleotide position 28 through nucleotide position 1923. The invention also discloses compositions and insecticidal formulations that comprise such a polypeptide. Such composition may be a cell extract, cell suspension, cell homogenate, cell lysate, cell supernatant, cell filtrate, or cell pellet of a bacteria cell that comprises a polynucleotide that encodes such a polypeptide. Exemplary bacterial cells that produce such a polypeptide include Bacillus thuringiensis EG4135 and EG4268, deposited with NRRL respectively on Apr. 28, 2000. The composition as described in detail below may be formulated as a powder, dust, pellet, granule, spray, emulsion, colloid, solution, or such like, and may be preparable by such conventional means as desiccation, lyophilization, homogenization, extraction, filtration, centrifugation, sedimentation, or concentration of a culture of cells comprising the polypeptide. Preferably such compositions are obtainable from cultures of Bacillus thuringiensis EG4135 and EG4268 cells. In all such compositions that contain at least one such insecticidal polypeptide, the polypeptide may be present in a concentration of from about 0.001% to about 99% by weight.
An exemplary insecticidal polypeptide formulation may be prepared by a process comprising the steps of culturing Bacillus thuringiensis EG4135 and EG4268 cells under conditions effective to produce the insecticidal polypeptide; and obtaining the insecticidal polypeptide so produced.
For example, the invention discloses and claims a method of preparing a xcex4-endotoxin polypeptide having insecticidal activity against a coleopteran insect. The method generally involves isolating from a culture of Bacillus thuringiensis EG4135 and EG4268 cells that have been grown under appropriate conditions, the xcex4-endotoxin polypeptide produced by the cells. Such polypeptides may be isolated from the cell culture or supernatant or from spore suspensions derived from the cell culture and used in the native form, or may be otherwise purified or concentrated as appropriate for the particular application.
A method of controlling a coleopteran insect population is also provided by the invention. The method generally involves contacting the population with an insecticidally-effective amount of a polypeptide comprising the amino acid sequence of SEQ ID NO:8. Such methods may be used to kill or reduce the numbers of coleopteran insects in a given area, or may be prophylactically applied to an environmental area to prevent infestation by a susceptible insect. Preferably the insect ingests, or is contacted with, an insecticidally-effective amount of the polypeptide.
Additionally, the invention provides a purified antibody that specifically binds to the insecticidal polypeptide. Also provided are methods of preparing such an antibody, and methods for using the antibody to isolate, identify, characterize, and/or purify polypeptides to which such an antibody specifically binds. Immunological kits and immunodetection methods useful in the identification of such polypeptides and peptide fragments and/or epitopes thereof are provided in detail herein, and also represent important aspects of the present invention.
Such antibodies may be used to detect the presence of such polypeptides in a sample, or may be used as described hereinbelow in a variety of immunological methods. An exemplary method for detecting a xcex4-endotoxin polypeptide in a biological sample generally involves obtaining a biological sample suspected of containing a xcex4-endotoxin polypeptide; contacting the sample with an antibody that specifically binds to the polypeptide, under conditions effective to allow the formation of complexes; and detecting the complexes so formed.
For such methods, the invention also provides an immunodetection kit. Such a kit generally contains, in suitable container means, an antibody that binds to the xcex4-endotoxin polypeptide, and at least a first immunodetection reagent. Optionally, the kit may provide additional reagents or instructions for using the antibody in the detection of xcex4-endotoxin polypeptides in a sample.
Preparation of such antibodies may be achieved using the disclosed polypeptide as an antigen in an animal as described below. Antigenic epitopes, shorter peptides, peptide fusions, carrier-linked peptide fragments, and the like may also be generated from a whole or a portion of the polypeptide sequence disclosed in SEQ ID NO:8. Particularly preferred peptides are those that comprise at least 10 contiguous amino acids from the sequence disclosed in SEQ ID NO:8.
In another embodiment, the present invention also provides nucleic acid segments that comprise a selected nucleotide sequence region that comprises the polynucleotide sequence of SEQ ID NO:7. In preferred embodiments, this selected nucleotide sequence region comprises a gene that encodes a polypeptide comprising at least SEQ ID NO:8.
Another aspect of the invention relates to a biologically-pure culture of a wild-type B. thuringiensis bacterium selected from the strains EG4135 and EG4268, deposited on Apr. 28, 2000 with the Agricultural Research Culture Collection, Northern Regional Research Laboratory (NRRL), Peoria, Ill. Also deposited was strain sIC8501 which is an E. coli DH5a containing plasmid pIC17501 which contains at least the native B. thuringiensis strain EG4135 tIC851 coding sequence. These strains were deposited under the terms of the Budapest Treaty, and viability statements pursuant to International Receipt Form BP/4 were obtained. B. thuringiensis strains EG4135 and EG4268 are naturally-occurring strains that contain at least one sequence region encoding the 632 amino acid long polypeptide sequence in SEQ ID NO:8.
A further embodiment of the invention relates to a vector comprising a sequence region that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:8, a recombinant host cell transformed with such a recombinant vector, and biologically-pure cultures of recombinant bacteria transformed with a polynucleotide sequence that encodes the polypeptide disclosed in SEQ ID NO:8. Exemplary vectors, recombinant host cells, transgenic cell lines, and transgenic plants comprising at least a first sequence region that encodes a polypeptide comprising the sequence of SEQ ID NO:8 are described in detail herein.
The present invention also provides transformed host cells, embryonic plant tissue, plant calli, plantlets, and transgenic plants that comprise a selected sequence region that encodes the insecticidal polypeptide. Such cells are preferably prokaryotic or eukaryotic cells such as bacterial, fungal, or plant cells, with exemplary bacterial cells including Bacillus thuringiensis, Bacillus subtilis, Bacillus megaterium, Bacillus cereus, Escherichia, Salmonella, Agrobacterium or Pseudomonas cells.
The plants and plant host cells are preferably monocotyledonous or dicotyledonous plant cells such as corn, wheat, soybean, oat, cotton, rice, rye, sorghum, sugarcane, tomato, tobacco, kapok, flax, potato, barley, turf grass, pasture grass, berry, fruit, legume, vegetable, ornamental plant, shrub, cactus, succulent, and tree cell.
Transgenic plants of the present invention preferably have incorporated into their genome or transformed into their chloroplast or plastid genomes a selected polynucleotide (or xe2x80x9ctransgenexe2x80x9d), that comprises at least a first sequence region that encodes the insecticidal polypeptide of SEQ ID NO:8. Transgenic plants are also meant to comprise progeny (descendant, offspring, etc.) of any generation of such a transgenic plant. A seed of any generation of all such transgenic insect-resistant plants wherein said seed comprises a DNA sequence encoding the polypeptide of the present invention is also an important aspect of the invention.
Insect resistant, crossed fertile transgenic plants comprising a transgene that encodes the polypeptide of SEQ ID NO:8 may be prepared by a method that generally involves obtaining a fertile transgenic plant that contains a chromosomally incorporated transgene encoding the insecticidal polypeptide of SEQ ID NO:8; operably linked to a promoter active in the plant; crossing the fertile transgenic plant with a second plant lacking the transgene to obtain a third plant comprising the transgene; and backcrossing the third plant to obtain a backcrossed fertile plant. In such cases, the transgene may be inherited through a male parent or through a female parent. The second plant may be an inbred, and the third plant may be a hybrid.
Likewise, an insect resistant hybrid, transgenic plant may be prepared by a method that generally involves crossing a first and a second inbred plant, wherein one or both of the first and second inbred plants comprises a chromosomally incorporated transgene that encodes the polypeptide of SEQ ID NO:8 operably linked to a plant expressible promoter that expresses the transgene. In illustrative embodiments, the first and second inbred plants may be monocot plants selected from the group consisting of: corn, wheat, rice, barley, oats, rye, sorghum, turfgrass and sugarcane.
In related embodiment, the invention also provides a method of preparing an insect resistant plant. The method generally involves contacting a recipient plant cell with a DNA composition comprising at least a first transgene that encodes the polypeptide of SEQ ID NO:8 under conditions permitting the uptake of the DNA composition; selecting a recipient cell comprising a chromosomally incorporated transgene that encodes the polypeptide; regenerating a plant from the selected cell; and identifying a fertile transgenic plant that has enhanced insect resistance relative to the corresponding non-transformed plant.
A method of producing transgenic seed generally involves obtaining a fertile transgenic plant comprising a chromosomally integrated transgene that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:8, operably linked to a promoter that expresses the transgene in a plant; and growing the plant under appropriate conditions to produce the transgenic seed.
A method of producing progeny of any generation of an insect resistance-enhanced fertile transgenic plant is also provided by the invention. The method generally involves collecting transgenic seed from a transgenic plant comprising a chromosomally integrated transgene that encodes the polypeptide of SEQ ID NO:8, operably linked to a promoter that expresses the transgene in the plant; planting the collected transgenic seed; and growing the progeny transgenic plants from the seed.
These methods for creating transgenic plants, progeny and seed may involve contacting the plant cell with the DNA composition using one of the processes well-known for plant cell transformation such as rnicroprojectile bombardment, electroporation or Agrobacterium-mediated transformation.
An exemplary method disclosed herein provides for protecting a plant from cotton boll weevil infestation comprising providing to a boll weevil in its diet a plant transformed to express a protein toxic to said weevil wherein said protein is expressed in sufficient amounts to control boll weevil infestation and wherein said protein is selected from the group consisting of Cry22Aa, ET70, and tIC851. In a further embodiment of this method, a plant expressing two or more of these proteins for the purpose of reducing boll weevil infestation is contemplated, in particular for reducing the development of races of boll weevils resistant to any of these proteins.
These and other embodiments of the present invention will be apparent to those of skill in the art from the following examples and claims, having benefit of the teachings of the Specification herein.
2.1 tIC851 Polynucleotide Sequences
The present invention provides polynucleotide sequences that can be isolated from Bacillus thuringiensis strains, that are free from total genomic DNA, and that encode the novel insecticidal polypeptides and peptide fragments disclosed herein. The polynucleotides encoding these peptides and polypeptides may encode active insecticidal proteins, or peptide fragments, polypeptide subunits, functional domains, or the like of one or more tIC851 or tIC851-related crystal proteins, such as the polypeptide disclosed in SEQ ID NO:8. In addition the invention encompasses nucleic acid sequences which may be synthesized entirely in vitro using methods that are well-known to those of skill in the art which encode the novel tIC851 polypeptide, peptides, peptide fragments, subunits, or functional domains disclosed herein.
As used herein, the term xe2x80x9cnucleic acid sequencexe2x80x9d or xe2x80x9cpolynucleotidexe2x80x9d refers to a nucleic acid molecule that has been isolated free of the total genomic DNA or otherwise of a particular species. Therefore, a nucleic acid sequence or polynucleotide encoding an endotoxin polypeptide refers to a nucleic acid molecule that comprises at least a first crystal protein-encoding sequence yet is isolated away from, or purified free from, total genomic DNA of the species from which the nucleic acid sequence is obtained, which in the instant case is the genome of the Gram-positive bacterial genus, Bacillus, and in particular, the species of Bacillus known as B. thuringiensis. Included within the term xe2x80x9cnucleic acid sequencexe2x80x9d, are polynucleotide sequences and smaller fragments of such sequences, and also recombinant vectors, including, for example, plasmids, cosmids, phagemids, phage, virions, baculoviruses, artificial chromosomes, viruses, and the like. Accordingly, polynucleotide sequences that have between about 70% and about 80%, or more preferably between about 81% and about 90%, or even more preferably between about 91% and about 99% nucleic acid sequence identity or functional equivalence to the polynucleotide sequence of SEQ ID NO:7 will be sequences that are xe2x80x9cessentially as set forth in SEQ ID NO:7.xe2x80x9d Highly preferred sequences are those which are preferably from about 91% to about 100% identical or functionally equivalent to the nucleotide sequence of SEQ ID NO:7. Other preferred sequences that encode tIC851 or tIC851-related sequences are those which are from about 81% to about 90% identical or functionally equivalent to the polynucleotide sequence set forth in SEQ ID NO:7. Likewise, sequences that are from about 71% to about 80% identical or functionally equivalent to the polynucleotide sequence set forth in SEQ ID NO:7 are also contemplated to be useful in the practice of the present invention.
Similarly, a polynucleotide comprising an isolated, purified, or selected gene or sequence region refers to a polynucleotide which may include in addition to peptide encoding sequences, certain other elements such as, regulatory sequences, isolated substantially away from other naturally occurring genes or protein-encoding sequences. In this respect, the term xe2x80x9cgenexe2x80x9d is used for simplicity to refer to a functional protein-, or polypeptide-encoding unit. As will be understood by those in the art, this functional term includes both genomic sequences, operator sequences and smaller engineered gene segments that express, or may be adapted to express, proteins, polypeptides or peptides. In certain embodiments, a nucleic acid segment will comprise at least a first gene that encodes a polypeptide comprising the sequence of SEQ ID NO:8.
To permit expression of the gene, and translation of the mRNA into mature polypeptide, the nucleic acid sequence preferably also comprises at least a first promoter operably linked to the gene to express the insecticidal polypeptide in a host cell transformed with this nucleic acid sequence. The promoter may be an endogenous promoter, or alternatively, a heterologous promoter selected for its ability to promote expression of the gene in one or more particular cell types. For example, in the creation of transgenic plants and plant cells comprising a tIC851 gene, the heterologous promoter of choice is one that is plant-expressible, and in many instances, may preferably be a plant-expressible promoter that is tissue- or cell cycle-specific. The selection of plant-expressible promoters is well-known to those skilled in the art of plant transformation, and exemplary suitable promoters are described herein. In certain embodiments, the plant-expressible promoter may be selected from the group consisting of corn sucrose synthetase 1, corn alcohol dehydrogenase 1, corn light harvesting complex, corn heat shock protein, pea small subunit RuBP carboxylase, Ti plasmid mannopine synthase, Ti plasmid nopaline synthase, petunia chalcone isomerase, bean glycine rich protein 1, Potato patatin, lectin, CaMV 35S, and the S-E9 small subunit RuBP carboxylase promoter.
xe2x80x9cIsolated substantially away from other coding sequencesxe2x80x9d means that the gene of interest, in this case, a gene encoding a bacterial crystal protein, forms the significant part of the coding region of the DNA segment, and that the DNA segment does not contain large portions of naturally-occurring coding DNA, such as large chromosomal fragments or other functional genes or operon coding regions. Of course, this refers to the DNA segment as originally isolated, and does not exclude genes, recombinant genes, synthetic linkers, or coding regions later added to the segment by the hand of man.
It will also be understood that this invention is not limited to the particular nucleic acid sequences which encode peptides of the present invention, or which encode the amino acid sequence of SEQ ID NO:8, including the DNA sequence which is particularly disclosed in SEQ ID NO:7. Recombinant vectors and isolated DNA segments may therefore variously include the polypeptide-coding regions themselves, coding regions bearing selected alterations or modifications in the basic coding region, or they may encode larger polypeptides that nevertheless include these peptide-coding regions or may encode biologically functional equivalent proteins or peptides that have variant amino acids sequences.
The DNA sequences of the present invention encompass biologically-functional, equivalent peptides. Such sequences may arise as a consequence of codon degeneracy and functional equivalency that are known to occur naturally within nucleic acid sequences and the proteins thus encoded. Alternatively, functionally-equivalent proteins or peptides may be created via the application of recombinant DNA technology, in which changes in the protein structure may be engineered, based on considerations of the properties of the amino acids being exchanged. If desired, one may also prepare fusion proteins and peptides, e.g., where the peptide-coding regions are aligned within the same expression unit with other proteins or peptides having desired functions, such as for purification or immunodetection purposes (e.g., proteins that may be purified by affinity chromatography and enzyme label coding regions, respectively). Recombinant vectors form further aspects of the present invention. Particularly useful vectors are contemplated to be those vectors in which the coding portion of the DNA sequence, whether encoding a full-length insecticidal protein or smaller peptide, is positioned under the control of a promoter. The promoter may be in the form of the promoter that is naturally associated with a gene encoding peptides of the present invention, as may be obtained by isolating the 5xe2x80x2 non-coding sequences located upstream of the coding segment or exon, for example, using recombinant cloning and/or PCR(trademark) technology, in connection with the compositions disclosed herein. In many cases, the promoter may be the native tIC851 promoter, or alternatively, a heterologous promoter, such as those of bacterial origin (including promoters from other crystal proteins), fungal origin, viral, phage or phagemid origin (including promoters such as CaMV35, and its derivatives, T3, T7, xcex, and xcfx86 promoters and the like), or plant origin (including constitutive, inducible, and/or tissue-specific promoters and the like).
In other embodiments, it is contemplated that certain advantages will be gained by positioning the coding DNA sequence under the control of a recombinant, or heterologous, promoter. As used herein, a recombinant or heterologous promoter is intended to refer to a promoter that is not normally associated with a DNA sequence encoding a crystal protein or peptide in its natural environment. Such promoters may include promoters normally associated with other genes, and/or promoters isolated from any bacterial, viral, eukaryotic, or plant cell. Naturally, it will be important to employ a promoter that effectively directs the expression of the DNA segment in the cell type, organism, or even animal, chosen for expression. The use of promoter and cell type combinations for protein expression is generally known to those of skill in the art of molecular biology, for example, see Sambrook et al., 1989. The promoters employed may be constitutive, or inducible, and can be used under the appropriate conditions to direct high level expression of the introduced DNA sequence, such as is advantageous in the large-scale production of recombinant proteins or peptides. Appropriate promoter systems contemplated for use in high-level expression include, but are not limited to, the Pichia expression vector system (Pharmacia LKB Biotechnology).
In yet another aspect, the present invention provides methods for producing a transgenic plant that expresses a selected nucleic acid sequence comprising a sequence region that encodes the novel endotoxin polypeptides of the present invention. The process of producing transgenic plants is well-known in the art. In general, the method comprises transforming a suitable plant host cell with a DNA sequence that contains a promoter operatively linked to a coding region that encodes one or more tIC851 polypeptides. Such a coding region is generally operatively linked to at least a first transcription-terminating region, whereby the promoter is capable of driving the transcription of the coding region in the cell, and hence providing the cell the ability to produce the polypeptide in vivo. Alternatively, in instances where it is desirable to control, regulate, or decrease the amount of a particular recombinant crystal protein expressed in a particular transgenic cell, the invention also provides for the expression of crystal protein antisense mRNA. The use of antisense mRNA as a means of controlling or decreasing the amount of a given protein of interest in a cell is well-known in the art.
Another aspect of the invention comprises transgenic plants which express a gene, gene sequence, or sequence region that encodes at least one or more of the novel polypeptide compositions disclosed herein. As used herein, the term xe2x80x9ctransgenic plantxe2x80x9d is intended to refer to a plant that has incorporated DNA sequences, including but not limited to genes which are perhaps not normally present, DNA sequences not normally transcribed into RNA or translated into a protein (xe2x80x9cexpressedxe2x80x9d), or any other genes or DNA sequences which one desires to introduce into the non-transformed plant, such as genes which may normally be present in the non-transformed plant but which one desires to either genetically engineer or to have altered expression.
It is contemplated that in some instances the genome of a transgenic plant of the present invention will have been augmented through the stable introduction of one or more transgenes, either native, synthetically modified, or mutated, that encodes an insecticidal polypeptide that is identical to, or highly homologous to the polypeptide disclosed in SEQ ID NO:8. In some instances, more than one transgene will be incorporated into the genome of the transformed host plant cell. Such is the case when more than one crystal protein-encoding DNA sequence is incorporated into the genome of such a plant. In certain situations, it may be desirable to have one, two, three, four, or even more B. thuringiensis crystal proteins (either native or recombinantly-engineered) incorporated and stably expressed in the transformed transgenic plant. Alternatively, a second transgene may be introduced into the plant cell to confer additional phenotypic traits to the plant. Such transgenes may confer resistance to one or more insects, bacteria, fungi, viruses, nematodes, or other pathogens.
A preferred gene which may be introduced includes, for example, a crystal protein-encoding DNA sequence from bacterial origin, and particularly one or more of those described herein which are obtained from Bacillus spp. Highly preferred nucleic acid sequences are those obtained from B. thuringiensis, or any of those sequences which have been genetically engineered to decrease or increase the insecticidal activity of the crystal protein in such a transformed host cell.
Means for transforming a plant cell and the preparation of plant cells, and regeneration of a transgenic cell line from a transformed cell, cell culture, embryo, or callus tissue are well-known in the art, and are discussed herein. Vectors, (including plasmids, cosmids, phage, phagemids, baculovirus, viruses, virions, BACs [bacterial artificial chromosomes], YACs [yeast artificial chromosomes]) comprising at least a first nucleic acid segment encoding an insecticidal polypeptide for use in transforming such cells will, of course, generally comprise either the operons, genes, or gene-derived sequences of the present invention, either native, or synthetically-derived, and particularly those encoding the disclosed crystal proteins. These nucleic acid constructs can further include structures such as promoters, enhancers, polylinkers, introns, terminators, or even gene sequences which have positively- or negatively-regulating activity upon the cloned xcex4-endotoxin gene as desired. The DNA sequence or gene may encode either a native or modified crystal protein, which will be expressed in the resultant recombinant cells, and/or which will confer to a transgenic plant comprising such a segment, an improved phenotype (in this case, increased resistance to insect attack, infestation, or colonization).
The preparation of a transgenic plant that comprises at least one polynucleotide sequence encoding a tIC851 or tIC851-derived polypeptide for the purpose of increasing or enhancing the resistance of such a plant to attack by a target insect represents an important aspect of the invention. In particular, the inventors describe herein the preparation of insect-resistant monocotyledonous or dicotyledonous plants, by incorporating into such a plant, a transgenic DNA sequence encoding at least one tIC851 polypeptide toxic to a coleopteran insect.
In a related aspect, the present invention also encompasses a seed produced by the transformed plant, a progeny from such seed, and a seed produced by the progeny of the original transgenic plant, produced in accordance with the above process. Such progeny and seeds will have a crystal protein-encoding transgene stably incorporated into their genome, and such progeny plants will inherit the traits afforded by the introduction of a stable transgene in Mendelian fashion. All such transgenic plants having incorporated into their genome transgenic DNA sequences encoding one or more tIC851 crystal proteins or polypeptides are aspects of this invention. As well-known to those of skill in the art, a progeny of a plant is understood to mean any offspring or any descendant from such a plant.
2.3 Definitions
The following words and phrases have the meanings set forth below.
A, an: In keeping with long-standing patent tradition, xe2x80x9caxe2x80x9d or xe2x80x9canxe2x80x9d used throughout this disclosure is intended to mean xe2x80x9cone or more.xe2x80x9d
Comprising, comprises: In keeping with long-standing patent tradition, xe2x80x9ccomprisingxe2x80x9d and xe2x80x9ccomprisesxe2x80x9d used throughout this disclosure is intended to mean xe2x80x9cincluding, but not limited to.xe2x80x9d
Expression: The combination of intracellular processes, including at least transcription and often the subsequent translation of mRNA of a coding DNA molecule such as a structural gene to produce a polypeptide.
Promoter: A recognition site on a DNA sequence or group of DNA sequences that provide an expression control element for a structural gene or sequence to be transcribed and to which an RNA polymerase specifically binds and initiates RNA synthesis (transcription) of that gene or sequence to be transcribed.
Regeneration: The process of growing a plant from a plant cell (e.g., plant protoplast or explant).
Structural gene: A DNA sequence that encodes a messenger RNA which can be transcribed to produce a polypeptide.
Transformation: A process of introducing an exogenous DNA sequence (e.g., a vector, a recombinant DNA molecule) into a cell, protoplast, or organelle within a cell, in which that exogenous DNA is incorporated into DNA native to the cell, or is capable of autonomous replication within the cell.
Transformed cell: A cell whose genotype has been altered by the introduction of an exogenous DNA sequence into that cell.
Transgenic cell: Any cell derived from or regenerated from a transformed cell. Exemplary transgenic cells include plant calli derived from a transformed plant cell and particular cells such as leaf, root, stem, e.g., somatic cells, or reproductive (germ) cells obtained from a transgenic plant.
Transgenic plant: A plant or a progeny of any generation of the plant that was derived from a transformed plant cell or protoplast, wherein the plant nucleic acids contains an exogenous selected nucleic acid sequence region not originally present in a native, non-transgenic plant of the same variety. The terms xe2x80x9ctransgenic plantxe2x80x9d and xe2x80x9ctransformed plantxe2x80x9d have sometimes been used in the art as synonymous terms to define a plant whose native DNA has been altered to contain a heterologous DNA molecule. However, it is thought more scientifically correct to refer to a regenerated plant or callus obtained from a transformed plant cell or protoplast cells as being a transgenic plant. Preferably, transgenic plants of the present invention include those plants that comprise at least a first selected polynucleotide that encodes an insecticidal polypeptide. This selected polynucleotide is preferably a xcex4-endotoxin coding region (or gene) operably linked to at least a first promoter that expresses the coding region to produce the insecticidal polypeptide in the transgenic plant. Preferably, the transgenic plants of the present invention that produce the encoded polypeptide demonstrate a phenotype of improved resistance to target insect pests. Such transgenic plants, their progeny, descendants, and seed from any such generation are preferably insect resistant plants.
Vector: A nucleic acid molecule capable of replication in a host cell and/or to which another nucleic acid sequence can be operably linked so as to bring about replication of the attached segment. Plasmids, phage, phagemids, and cosmids are all exemplary vectors. In many embodiments, vectors are used as a vehicle to introduce one or more selected polynucleotides into a host cell, thereby generating a xe2x80x9ctransformedxe2x80x9d or xe2x80x9crecombinantxe2x80x9d host cell.